Molecular Cytology Research Articles

Highly Sensitive and Specific Molecular Test for Mutations in the Diagnosis of Thyroid Nodules: A Prospective Study of BRAF-Prevalent Population.

Molecular testing offers more objective information in the diagnosis and personalized decision making for thyroid nodules. In Korea, as the BRAF V600E mutation is detected in 70–80% of thyroid cancer specimens, its testing in fine-needle aspiration (FNA) cytology specimens alone has been used for the differential diagnosis of thyroid nodules until now. Thus, we aimed to develop a mutation panel to detect not only BRAF V600E, but also other common genetic alterations in thyroid cancer and to evaluate the diagnostic accuracy of the mutation panel for thyroid nodules in Korea. For this prospective study, FNA specimens of 430 nodules were obtained from patients who underwent thyroid surgery for thyroid nodules. A molecular test was devised using real-time PCR to detect common genetic alterations in thyroid cancer, including BRAF, N-, H-, and K-RAS mutations and rearrangements of RET/PTC and PAX8/PPARr. Positive results for the mutation panel were confirmed by sequencing. Among the 430 FNA specimens, genetic alterations were detected in 293 cases (68%). BRAF V600E (240 of 347 cases, 69%) was the most prevalent mutation in thyroid cancer. The RAS mutation was most prevalently detected for indeterminate cytology. Among the 293 mutation-positive cases, 287 (98%) were diagnosed as cancer. The combination of molecular testing and cytology improved sensitivity from 72% (cytology alone) to 89% (combination), with a specificity of 93%. We verified the excellent diagnostic performance of the mutation panel applicable for clinical practice in Korea. A plan has been devised to validate its performance using independent FNA specimens.

Infraspecific diversification of the star cloak fern (Notholaena standleyi) in the deserts of the United States and Mexico.

Not all ferns grow in moist and shaded habitats. One well-known example is Notholaena standleyi, a species that thrives in deserts of the southwestern United States and Mexico. This species exhibits several "chemotypes" that differ in farina (flavonoid exudates) color and chemistry. By integrating data from molecular phylogenetics, cytology, biochemistry, and biogeography, we circumscribed the major evolutionary lineages within N. standleyi and reconstructed their diversification histories. Forty-eight samples were selected from across the geographic distribution of N. standleyi. Phylogenetic relationships were inferred using four plastid and five nuclear markers. Ploidy levels were inferred using spore sizes calibrated by chromosome counts, and farina chemistry was compared using thin-layer chromatography. Four clades are recognized, three of which roughly correspond to previously recognized chemotypes. The diploid clades G and Y are found in the Sonoran and Chihuahuan deserts, respectively; they are estimated to have diverged in the Pleistocene, congruent with the postulated timing of climatological events separating these two deserts. Clade P/YG is tetraploid and partially overlaps the distribution of clade Y in the eastern Chihuahuan Desert. It is apparently confined to limestone, a geologic substrate rarely occupied by members of the other clades. The cryptic (C) clade, a diploid group known only from southern Mexico and highly disjunct from the other three clades, is newly recognized here. Our results reveal a complex intraspecific diversification history of N. standleyi, traceable to a variety of evolutionary drivers including classic allopatry, parapatry with or without changes in geologic substrate, and sympatric divergence through polyploidization.

The Impact of Global Transcriptional Regulation on Bacterial Gene Order.

SummaryBacterial gene expression depends on the allocation of limited transcriptional resources provided a particular growth rate and growth condition. Early studies in a few genes suggested this global regulation to generate a unifying hyperbolic expression pattern. Here, we developed a large-scale method that generalizes these experiments to quantify the response to growth of over 700 genes that a priori do not exhibit any specific control. We distinguish a core subset following a promoter-specific hyperbolic response. Within this group, we sort genes with regard to their responsiveness to the global regulatory program to show that those with a particularly sensitive linear response are located near the origin of replication. We then find evidence that this genomic architecture is biologically significant by examining position conservation of E. coli genes in 100 bacteria. The response to the transcriptional resources of the cell results in an additional feature contributing to bacterial genome organization.

Processing of Fine-Needle Aspiration Specimens from Pancreatic Lesions.

It is important to adequately process and triage the specimen obtained from fine-needle aspirations (FNAs) of pancreatic lesions. Many echo endoscopists rely on rapid on-site evaluation (ROSE) for adequacy of FNA from solid pancreatic lesions. The role of ROSE in FNA of pancreatic lesions is discussed, as is the triage of material for making smears and cell block preparation. Different techniques of cell block preparation are briefly mentioned. Pancreatic cystic fluid obtained from pancreatic cysts is triaged differently as compared to specimens obtained from solid pancreatic lesions. An algorithmic approach to the processing of pancreatic cystic fluid for molecular and biochemical assays and cytology is discussed. Proper specimen handling is crucial to the accurate interpretation of pancreatic FNA specimens. The methods used to process a sample depend on whether the aspirated sample is solid or cystic and the type of device used for sampling. ROSE has been shown to reduce the number of inadequate specimens and to improve specimen preparation. The details of the various cytological preparation methods available are described in numerous texts. Here we focus on providing a broad overview of specimen collection and processing as it relates to pancreatic FNA, with guidance to the reader based on published and personal experiences.

3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop.

Phylogeny and maternal donors of Elytrigia Desv. sensu lato (Triticeae; Poaceae) inferred from nuclear internal-transcribed spacer and trnL-F sequences

BackgroundElytrigia Desv. is a genus with a varied array of morphology, cytology, ecology, and distribution in Triticeae. Classification and systematic position of Elytrigia remain controversial. We used nuclear internal-transcribed spacer (nrITS) sequences and chloroplast trnL-F region to study the relationships of phylogenetic and maternal genome donor of Elytrigia Desv. sensu lato.Results(1) E, F, P, St, and W genomes bear close relationship with one another and are distant from H and Ns genomes. Ee and Eb are homoeologous. (2) In ESt genome species, E genome is the origin of diploid Elytrigia species with E genome, St genome is the origin of Pseudoroegneria. (3) Diploid species Et. elongata were differentiated. (4) Et. stipifolia and Et. varnensis sequences are diverse based on nrITS data. (5) Et. lolioides contains St and H genomes and belongs to Elymus s. l. (6) E genome diploid species in Elytrigia serve as maternal donors of E genome for Et. nodosa (PI547344), Et. farcta, Et. pontica, Et. pycnantha, Et. scirpea, and Et. scythica. At least two species act as maternal donor of allopolyploids (ESt and EStP genomes).ConclusionsOur results suggested that Elytrigia s. l. species contain different genomes, which should be divided into different genera. However, the genomes of Elytrigia species had close relationships with one another. Diploid species were differentiated, because of introgression and different geographical sources. The results also suggested that the same species and the same genomes of different species have different maternal donor. Further study of molecular biology and cytology could facilitate the evaluation of our results of phylogenetic in a more specific and accurate way.

Molecular phylogeny reveals the non-monophyly of tribe Yinshanieae (Brassicaceae) and description of a new tribe, Hillielleae

The taxonomic treatment within the unigeneric tribe Yinshanieae (Brassicaceae) is controversial, owing to differences in generic delimitation applied to its species. In this study, sequences from nuclear ITS and chloroplast trnL-F regions were used to test the monophyly of Yinshanieae, while two nuclear markers (ITS, ETS) and four chloroplast markers (trnL-F, trnH-psbA, rps16, rpL32-trnL) were used to elucidate the phylogenetic relationships within the tribe. Using maximum parsimony, maximum likelihood, and Bayesian inference methods, we reconstructed the phylogeny of Brassicaceae and Yinshanieae. The results show that Yinshanieae is not a monophyletic group, with the taxa splitting into two distantly related clades: one clade contains four taxa and falls in Lineage I, whereas the other includes all species previously placed in Hilliella and is embedded in the Expanded Lineage II. The tribe Yinshanieae is redefined, and a new tribe, Hillielleae, is proposed based on combined evidence from molecular phylogeny, morphology, and cytology.

Targeted next generation sequencing of endoscopic ultrasound acquired cytology from ampullary and pancreatic adenocarcinoma has the potential to aid patient stratification for optimal therapy selection.

Background & AimsLess than 10% of registered drug intervention trials for pancreatic ductal adenocarcinoma (PDAC) include a biomarker stratification strategy. The ability to identify distinct mutation subsets via endoscopic ultrasound fine needle aspiration (EUS FNA) molecular cytology could greatly aid clinical trial patient stratification and offer predictive markers. We identified chemotherapy treatment naïve ampullary adenocarcinoma and PDAC patients who underwent EUS FNA to assess multigene mutational frequency and diversity with a surgical resection concordance assessment, where available.MethodsFollowing strict cytology smear screening criteria, targeted next generation sequencing (NGS) using a 160 cancer gene panel was performed.ResultsComplete sequencing was achieved in 29 patients, whereby 83 pathogenic alterations were identified in 21 genes. Cytology genotyping revealed that the majority of mutations were identified in KRAS (93%), TP53 (72%), SMAD4 (31%), and GNAS (10%). There was 100% concordance for the following pathogenic alterations: KRAS, TP53, SMAD4, KMT2D, NOTCH2, MSH2, RB1, SMARCA4, PPP2R1A, PIK3R1, SCL7A8, ATM, and FANCD2. Absolute multigene mutational concordance was 83%. Incremental cytology smear mutations in GRIN2A, GATA3 and KDM6A were identified despite re-examination of raw sequence reads in the corresponding resection specimens.ConclusionsEUS FNA cytology genotyping using a 160 cancer gene NGS panel revealed a broad spectrum of pathogenic alterations. The fidelity of cytology genotyping to that of paired surgical resection specimens suggests that EUS FNA represents a suitable surrogate and may complement the conventional stratification criteria in decision making for therapies and may guide future biomarker driven therapeutic development.

Molecular cytology genotyping of primary and metastatic GIstromal tumors by using a custom two-gene targeted next-generation sequencing panel with therapeutic intent.

In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens.

Sa1455 KRAS Mutant Subtype Assessment of Pancreatic and Ampullary Adenocarcinoma by EUS FNA Molecular Cytology Targeted Next Generation Sequencing

Sa1455 KRAS Mutant Subtype Assessment of Pancreatic and Ampullary Adenocarcinoma by EUS FNA Molecular Cytology Targeted Next Generation Sequencing

Sa1355 Molecular Cytology Genotyping of Primary and Metastatic Gastrointestinal Stromal Tumors Using Targeted Next Generation Sequencing to Guide Therapeutic Decision Making

Sa1355 Molecular Cytology Genotyping of Primary and Metastatic Gastrointestinal Stromal Tumors Using Targeted Next Generation Sequencing to Guide Therapeutic Decision Making

Micrometastases identification in malignant tumors

The article reviewed literature data relating to the methods used for detection of single tumor cells in bone marrow, lymph nodes, and peripheral blood. Sensitivity of modern detection methods is analyzed. Despite advances in the development of molecular biology and cytology, until now there is no universal approach to the micrometastases identification, and existing methods optimization are recommended.

An update on pleuro‐pulmonary cytopathology: Part i: Cytological diagnosis of mesothelioma and molecular cytology of lung cancer with an historical perspective

An update on pleuro‐pulmonary cytopathology: Part i: Cytological diagnosis of mesothelioma and molecular cytology of lung cancer with an historical perspective

Phylogenetic analyses reveal three new genera of the Campanulaceae

AbstractThe genus Codonopsis has been controversial in its circumscription. In the strict sense, the genus consists of three subgenera, Codonopsis, Pseudocodonopsis, and Obconicicapsula. Recent molecular and palynological analyses suggest that the latter two subgenera and Cod. purpurea Wall. are separated from rest Codonopsis. In the present study, extensive observations were carried out in wild populations and on more than 800 specimens of subg. Pseudocodonopsis, subg. Obconicicapsula, and Cod. purpurea. Their chromosome numbers were counted for the first time. Recent palynological and molecular data were also analyzed and reevaluated. Our integrated phylogenetic analyses revealed that the three groups are distinct from the rest of Codonopsis. Furthermore, they are also distinct from each other, and cannot be circumscribed in a single genus. Therefore, three new genera are here proposed based on molecular phylogenetics, palynology, cytology, and external morphology. New combinations are made in the present article.

Improving the accuracy of pancreatobiliary tract cytology with fluorescence in situ hybridization

The detection of aneuploidy by fluorescence in situ hybridization (FISH) has revolutionized how laboratories diagnose cholangiocarcinoma and pancreatic adenocarcinoma using cytology specimens. Numerous clinical studies have demonstrated that FISH increases the diagnostic sensitivity of routine cytology for detecting pancreatobiliary tract malignancy with minimal decreases in clinical specificity. FISH also provides useful information in difficult clinical scenarios, including the assessment of patients with biliary strictures who have equivocal cytology results and the assessment of patients with primary sclerosing cholangitis who have clinical features suggestive of malignancy. The improved ability to detect pancreatobiliary tract cancers offers the possibility of earlier detection when patients are amenable to surgical intervention and can decrease health care costs by reducing the amount of clinical evaluation required to arrive at a cancer diagnosis. Cytopathology personnel should maintain familiarity with molecular cytology testing methodologies, because morphologic and aneuploidy assessment of tumors will continue to be an integral part of large-scale genome analyses of individual tumors.

MRNA and DNA Detection of Human Papillomaviruses in Women of All Ages Attending Two Colposcopy Clinics

ObjectiveHPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals.Methods1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results.ResultsHPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests’ positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used.ConclusionsOverall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results.

Molecular diagnosis on tissues and cells: how it affects training and will affect practice in the future

On 25th November 2011, a symposium organized by the Royal College of Pathologists, entitled 'Molecular diagnosis on tissues and cells', took place in London. As trainees in histopathology and cytopathology, we were stimulated to consider the role that molecular biology is likely to play in future practice and how this is addressed by our own training. The symposium provided a basis for this article. Routine samples requiring molecular analysis are equally relevant to histopathologists and cytopathologists, and molecular biology laboratories are now using cytological as well as histological material for diagnostic testing, allowing different specimen types to be used as and when they are most appropriate. The most widely used types of molecular analysis in routine cellular pathology are EGFR testing in lung cancer, molecular testing of thyroid nodules, fluorescence in situ hybridization testing of urine samples, clonality analysis in lymphoma testing, HER2 testing in breast and gastric cancer, KRAS testing in colorectal cancer, intraoperative assessment of breast cancer sentinel nodes, molecular testing of gastrointestinal stromal tumours and mismatch repair protein analysis. Of these, the majority in the UK are carried out on histopathology samples, although many are applicable to cytological samples if adequate material is obtained. We are particularly encouraged by the potential of molecular diagnostic cytology in traditionally difficult areas, such as intraoperative assessment. We believe that increasing reliance on molecular diagnostic techniques will also herald changes in training.

Identification and molecular cytology analysis of cold tolerance introgression lines derived from Oryza sativa L. mating with O. rufipogon Griff.

A previously constructed introgression lines (ILs) population including 239 lines was used to evaluate the cold-tolerant ability. The ILs with strong cold tolerance which were identified in the present study were used for further cytological and microsatellite (SSR) marker analyses. The results showed that the IL5243 and IL5335 had strong cold-tolerant ability. Cytological analysis showed that the rate of pollen mother cells (PMCs) with normal meiotic behavior in the IL5243 and IL5335 was to 89.93 and 90.22 %, respectively, and finally formed normal mature pollen. And meanwhile, the low frequency of abnormal chromosome behavior was observed in the IL5243 and IL5335, such as univalent, 8-shape bivalent, multivalent at diakinesis. At anaphase I, one or two lagging chromosomes were observed in some PMCs (3.95–5.15 %). The results of SSR marker analysis further confirmed that partial alien DNA of common wild rice has been transferred into the IL5243 and IL5335. These results implied that the IL5243 and IL5335 might be excellent bridging germplasm for exploring and utilizing the cold-tolerant gene of common wild rice. In addition, IL5243 and IL5335 would provide a better experimental system for understanding some epigenetic phenomenon induced by alien gene introgression.

Abstract A36: Identification of chromatin-based patterns across lung cancer grades

Abstract A hallmark of the transformed phenotype is altered chromosomal structure. The development and progression of lung cancer, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes. Although there have been numerous studies on chromosomal aberrations in lung cancer, and cancer in general, broad assessment of chromatin structure information has been understudied, and its role in malignant transformation remains poorly characterized. Cancer progression is classified by tumor grade, which is determined by examining morphological changes that cells undergo as they de-differentiate. Given that chromosomal aberrations are well-documented in nearly all cancers, it is surprising that there is currently no information on the role of chromatin structure in the progression of cancer. We have identified chromatin-based patterns across different lung adenocarcinoma cancer grades. To address the role of chromatin structure in the progression of cancer, we compared the chromatin structure from primary lung adenocarcinomas of grades one, two and three to their normal adjacent tissue, from multiple individuals, using an innovative microarray strategy. We measured chromatin structure across cancer grades between normal and tumor samples at three levels of resolution: nucleosome distribution, chromatin accessibility and three-dimensional molecular cytology. We used micrococcal nuclease (MNase) as a surrogate for highly diffusible molecules such as transcription factors, mobile elements, replication and repair machinery, and small RNAs. Our work indicates that grade one lung adenocarcinomas have greatly altered nucleosome distributions compared to the adjacent normal tissue, but nearly identical chromatin accessibility. Conversely, the grade three samples show massive disruptions in chromosomal accessibility, but only modest changes in nucleosome distribution when tumor and normal samples are compared. This data has allowed us to develop a model in which we propose that early grade lung adenocarcinomas are linked to changes in nucleosome distributions, while later grade cancers are linked to large-scale chromosomal changes. These results indicate that we should be able to use these chromatin structural changes to identify grade sub-type specific cancer biomarkers. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A36.

Current State and Future Perspective of Molecular Diagnosis of Fine-Needle Aspiration Biopsy of Thyroid Nodules

Fine-needle aspiration biopsy (FNAB) is the most sensitive and specific tool for the differential diagnosis of thyroid malignancy. Some limitations of FNAB can be overcome by the molecular analysis of FNAB. This review analyzes the current state and problems of the molecular analysis of FNAB as well as possible goals for increasing the diagnostic rate, especially in the indeterminate/follicular lesion cytological group. Twenty publications were evaluated for the diagnostic material and assay systems used, the type, and the number of mutations screened. Sensitivity, specificity, and false-negative and false-positive rates were calculated for all publications. Testing for a panel of somatic mutations is most promising to reduce the number of indeterminate FNAB. A mean sensitivity of 63.7% was achieved for indeterminate lesions. However, there is a broad sensitivity range for the investigation of mutations in the indeterminate lesions. Therefore, additional molecular markers should be defined by mRNA and microRNA expression studies and evaluated in FNAB samples of thyroid carcinomas without known somatic mutations, and especially for the many benign nodules in the indeterminate/follicular lesion fine-needle aspiration cytology category. This approach should improve the differential diagnosis of indeterminate/follicular lesion FNAB samples. Testing for a panel of somatic mutations has led to an improvement of sensitivity/specificity for indeterminate/follicular proliferation FNAB samples. Further methodological improvements, standardizations, and further molecular markers should soon lead to a broader application of molecular FNAB cytology for the differential diagnosis of thyroid nodules and to a substantial reduction of diagnostic surgeries.